Pertussis toxin is the major virulence factor of Bordetella pertussis. It is the most complex toxin known. Five different subunits associate in an unusual, 1:1:1:2:1 ratio. The subunits are secreted to the periplasm by the Sec-pathway where intramolecular disulfide bonds form and the subunits assemble into the functional toxin. Nine Ptl (for pertussis toxin liberation) proteins mediate secretion of properly assembled toxin past the outer membrane. Specific Aim 1. Characterization of periplasmic folding and assembly of pertussis toxin. We have shown that disulfide bond formation occurs by an unusual pathway in Bordetella pertussis. The role of the novel periplasmic folding chaperones, and small thiols in the assembly of pertussis toxin will be investigated. Specific Aim 2. The role of individual Ptl proteins in secretion. The function of the nine Ptl proteins will be investigated using genetic and biochemical methods with the goals of identifying the channel forming proteins, the glycohydrolase, proteins involved in assembly of the secretion complex, and proteins involved in specificity (e.g. distinguishing individual subunits from assembled pertussis toxin). Specific Aim 3. Horizontal transfer of pertussis toxin genes. Two non-functional pertussis toxin operons (the chromosomal operon in Bordetella bronchiseptica, and the phage-encoded operon in Bordetella avium) will be investigated to gain an understanding of the unique events that occurred in the species B. pertussis that permit only them to express pertussis toxin. It is our hope that a detailed molecular understanding of the pertussis toxin secretion process will lead to better treatments for toxigenic diseases, including new therapeutics to block secretion, or "anti-virulence" therapies that could be as effective as the current anti-microbial therapies.